Much evidence has accumulated supporting the hypothesis that the purine nucleoside adenosine may indeed function as a neuromodulator in the mammalian retina, but to date no reports have directly illustrated a physiological role for this nucleoside. In other regions of the CNS, adenosine agonists decrease transmitter release, whereas antagonists increase release. A similar role for adenosine in the retina is now apparent. The cholinergic amacrine cells of the rabbit retina were labeled with [3H]choline, and the effects of enzymatic adenosine degradation or adenosine antagonists on the light-evoked efflux of acetylcholine were evaluated. When endogenous adenosine was degraded by addition of adenosine deaminase, the light-evoked release of radioactivity derived from [3H]choline was significantly increased compared with control values. A similar response was observed when rabbit eyecups were superfused with a selective adenosine A1 receptor antagonist. The effect elicited by adenosine deaminase could be almost completely reversed by addition of cyclopentyladenosine, a highly selective A1 receptor agonist. These effects were observed in either the presence or the absence of picrotoxin. The results demonstrate a modulation of retinal physiology by adenosine.